Gene activation requires interaction of DNA-bound activators with proteins binding near the transcription start site of a gene (Ptashne, Nature 335:983, 1988). In eukaryotes, activation of RNA polymerase II genes requires many transcription factors in addition to RNA polymerase. Transcriptional activators have been shown to contact one or another of these transcription factors, including TATA-binding protein (TBP), TBP-associated factors (TAFs), TFIIB, and TFIIH (Roeder, Trends Biochem. Sci. 16:402, 1991; Zawel et al., Prog. Nucl. Acids Res. Mol. Biol. 44:67, 1993; Conaway et al., Annu. Rev. Biochem. 62:161, 1993; Hoey et al., Cell 72:247). Thus, it has been proposed that transcription initiation involves a multistep assembly process, various steps of which might be catalyzed by activators (Buratowski et al., Cell 56:549, 1989; Choy et al., Nature 366:531, 1993).
Some transcriptional activators are thought to recruit one or more transcription factors to the DNA, to cause crucial conformational changes in target proteins and thereby to facilitate the complex process of assembling the transcriptional machinery, or both (Lin et al., Cell 64:971, 1991; Roberts et al., Nature 371:717, 1994; Hori et al., Curr. Op. Genet. Dev. 4:236, 1994). Also, given the observation that yeast RNA polymerase II is associated with several transcription factors, in a complex termed the “holoenzyme”, it has been proposed that some transcriptional activators might function by recruiting the holoenzyme complex to DNA (Koleske et al., Nature 368:466, 1994; Kim et al., Cell 77:599, 1994; Carey, Nature 368:402, 1994).
Transcriptional activation has been much studied both in the context of controlling gene expression in cells, for example so that principles of gene activation can be employed in genetic therapies, and as an experimental tool for analysis of protein—protein interactions in cells (Fields et al., Nature 340:245, 1989; Gyuris et al., Cell 75:791, 1993). One difficulty that has been encountered in the use and analysis of transcriptional activation systems, however, is that over-expression of transcriptional activators in cells typically inhibits gene expression, sometimes with dire results on the cells. This effect, termed “squelching”, apparently represents the titration of a transcription factor by the over-expressed transcriptional activator (Gill et al., Nature 334:721, 1988). Another difficulty that has been encountered specifically in the protein—protein interaction applications is that useful controls are often unavailable, so that spurious results are often observed. Also, the protein—protein interaction systems are typically not useful for identification of proteins that interact with transcriptional activators themselves. Given that transcriptional activators represent a significant fraction of all known proteins, this limitation of existing systems presents a serious problem.
There remains a need for the identification of novel transcriptional activators and improved transcriptional activation systems. In particular, there is a need for strong transcriptional activators that do not “squelch” other known activators, and for protein—protein interaction systems useful for identifying interaction partners of transcriptional activators.